CN112292368B - Synthesis of (+) -cannabinoids and therapeutic uses thereof - Google Patents

Abstract

The present invention relates to a process for the production of a compound of formula (I) or a salt of a compound of formula (I). The invention also relates to a compound of formula (I) or a salt of a compound of formula (I), for use in a method of treatment for achieving one or more therapeutic effects, and for use in the treatment and/or prophylaxis of certain diseases. Furthermore, the present invention provides a pharmaceutical composition comprising one or more compounds of formula (I) or one or more salts of compounds of formula (I).

Description

Synthesis of (+) -cannabinoids and therapeutic uses thereof

Technical Field

The present invention relates to a process for the production of a compound of formula (I)

Or a salt of a compound of formula (I). The meanings of X and n are explained below and preferred compounds are identified in the disclosure below. The invention also relates to compounds of formula (I) or salts of compounds of formula (I), methods of treatment for achieving one or more therapeutic effects, and for treating and/or preventing certain diseases. Furthermore, the present invention provides a pharmaceutical composition comprising one or more compounds of formula (I) or one or more salts of compounds of formula (I).

Background

The most common cannabinoid in cannabis, cannabidiol (CBD), is of increasing interest for medical use due to its broad spectrum of biological activity. CBD is used as an effective pharmaceutical ingredient in combination with dronabinol for MS treatment and as a single drug for the treatment of epileptic disorders like Dravet syndrome. In various clinical trials, the anti-epileptic therapeutic effect of the structurally related compound Cannabidiol (CBDV) was investigated.

Naturally occurring CBD and CBDV have the absolute steric configuration (-) -trans. The cis-isomer or (+) -enantiomer is not produced in plants and thus has no pharmaceutical effect so far. The first biological study of the enantiomer (+) -trans-CBD showed surprising biological effects (Morales et al, "pharmaceutical chemical overview of synthetic and natural derivatives of cannabidiol (An overview on medicinal chemistry of synthetic and natural derivatives of cannabidiol)", pharmacological front (Frontiers in Pharmacology), 2017,Vol.8,Article 422,doi:10.3389/fphar.2017.00422). However, existing biological data is not mature.

In the legalization of cannabinoids as pharmaceutical products, the demand for CBD is increasing, which has led to the development of alternatives for cannabis plant cultivation. In addition to the various biosynthetic pathways that are still currently under development, chemical synthesis of cannabinoids is the most promising option. Chemical synthesis can produce a pharmaceutically effective component that is safe and effective to administer. However, every synthetic process of chiral molecules like CED must prove that the product is chirally pure. The cis configuration may be excluded due to chemical processes (such as CBD processes in EP2842933B 1). To prove that the product is enantiomerically pure, in the case of CBD this would involve the (-) -configuration, a procedure requiring either a confirmed enantiomerically pure primary product or a defined chiral analysis method. These methods may be methods for measuring the optical rotation of the product, and may be chiral chromatography such as HPLC or GC. However, to verify these methods, both enantiomers are necessary. Thus, (+) -cannabinoids also have great analytical potential as reference substances.

WO 2017/011440 A1 relates to the synthesis of (+) -cannabidiol using halogens and halogen-containing compounds (bromide, dichloromethane) and requiring a huge instrument complexity. US2017/0349517A1 discloses the synthesis of (+) -hypocreol, which is carried out in a batch process using methylene chloride as solvent and requiring cooling to-10 ℃ to-15 ℃.

There is still a need for efficient methods to produce enantiomerically pure (+) -cannabinoids and to identify compounds with biological activity that allow therapeutic and/or prophylactic medical applications.

Disclosure of Invention

It is therefore an object of the present invention to provide a process for the production of enantiomerically pure (+) -cannabinoids. In particular, the synthesis should be efficient and allow for a continuous synthesis process without requiring significant instrument complexity. Furthermore, it is an object of the present invention that the purification of the product should be easy and in particular that no (chiral) chromatography step is required. Furthermore, it should be possible to start from diastereomerically pure educts.

It is a further object of the present invention to provide enantiomerically pure (+) -cannabinoids which are biologically active, useful for achieving a specific therapeutic effect and allowing the treatment and/or prophylaxis of certain diseases.

By a process for the production of compounds of formula (I)

Or a salt of a compound of formula (I),

wherein x=h or-COOY,

wherein Y = a saturated or unsaturated, branched or unbranched alkyl, aryl or heteroaryl group, each having 1 to 12 carbon atoms, and being optionally substituted by one or more amino groups, hydroxyl groups and/or halogens, and

where n=2 or 4,

the method comprises the following steps:

i) Reacting 4S-menthadienol with a compound of formula (II),

where n=2 or 4,

to obtain a compound of formula (III),

where n=2 or 4.

Detailed Description

The first step of the process is the Friedel Craft addition reaction of 4S-menthol and a substituted resorcinol derivative (methyl olivate or divarin methyl ester) over an acid catalyst. This step does not require cooling and is therefore preferably carried out between 20 ℃ and 25 ℃. To ensure the (+) -configuration of each cannabinoid methyl ester, the starting material, menthadienol, must have a 4S configuration. Since the 1S/1R stereocenter is destroyed during the addition, it may be 1S, 4S-menthadienol, 1R, 4S-menthadienol, or a mixture of both menthadienols. On the other hand, 4R-menthol impurities may result in the respective (-) -cannabinoid. To examine the stability of the process and its purification, a 4S-menthadienol starting material containing 5% of 4R-menthadienol as an impurity can be used.

Scheme 1: friedel Craft addition of 4S-menthadienol with substituted resorcinol derivatives

According to one embodiment, the above method further comprises one or more steps of:

ii) transesterification of the compounds of formula (III), and/or

iii) Decarboxylation of the compound of formula (III).

No purification of the primary product, i.e. the compound of formula (III), is required before step ii) is carried out. Step ii) of the process involves transesterification of the (+) -cannabinoid methyl ester followed by acidic decarboxylation to the desired (+) -cannabinoid. The transesterification is preferably carried out in vacuo, preferably between 300mbar and 700mbar, more preferably between 400mbar and 600mbar, particularly preferably about 500mbar, to allow immediate distillation of the low-boiling ethanol. The intermediate esters formed by the transesterification reaction are generally not isolated. Any alkyl alcohol may be used as a reaction partner in the transesterification reaction. Preferred alcohols are given below. The product does not require chiral chromatographic purification to achieve > 99% chiral purity.

According to a preferred embodiment, in the above process, step i) is carried out as a batch or, preferably, as a continuous flow reaction process.

The first step may be performed as a batch reaction, but better yields and purities may be obtained if the flow cell reactor is used as a continuous flow reaction.

Table 1: comparison of batch-continuous flow reactions for the Synthesis of (+) -trans-cannabidiol methyl ester (CBD-ME)

Reaction type	Batch size	Purity primary CBD-ME	Yield is good
Batch reaction	50g	57％	46％
					100g	52％	46％
				Continuous flow reaction	50g	78％	68％
	100g	79％	71％

In another preferred embodiment of the above method, the compound of formula (I) is selected from the group consisting of compounds of formulas (1) to (5) or salts thereof

Compound (1): (+) -cannabidiol methyl ester ((+) -CBD-ME)

Compound (2): (+) -cannabidiol ethylene glycol ester ((+) -CBD-GE)

Compound (3): (+) -cannabidiol ((+) -CBD)

Compound (4): (+) -cannabidiol hydroxypentyl ester ((+) -CBD-HPE)

Compound (5): (+) -cannabidiol ((+) -CBDV)

Compounds (1) to (5) have been shown to be biologically active, allowing therapeutic applications as explained in more detail below.

For the compounds of formula (I) and the salts of compounds (1) to (5) in the context of the present invention, the following applies: where appropriate, one or more hydroxyl groups of one or more compounds are present in deprotonated form. In addition to one or more (deprotonated) compounds, a corresponding number of counter cations are present, wherein these cations are preferably selected from the group consisting of: single positive charge cations of the first main group and the first group transition elements, ammonium ions, trialkylammonium ions, double positive charge cations of the second main group and the second group transition elements, and triple positive charge cations of the third main group and the third group transition elements, and combinations thereof.

The phenolic hydroxyl groups of the compounds are generally more acidic than the hydroxyl groups in the aliphatic side chains (if present).

The corresponding number of counter cations (depending on their charge) comes from the number of deprotonated hydroxyl groups. For example, itThe presence of a doubly negatively charged anion in the case of compounds of formula (I) having two phenolic hydroxyl groups as the basis for this salt, in which case these phenolic hydroxyl groups are completely deprotonated, leads to the number of positive charges (in this case: two) which have to be provided by one or more counter cations. Most preferably, these counter cations are selected from Na ⁺ 、K ⁺ 、NH ₄ ⁺ 、Ca ²⁺ 、Mg ²⁺ 、Al ³⁺ And Zn ²⁺ A group of groups.

Further preferred is the above process wherein in step i) pure 1S, 4S-menthadienol or pure 1S, 4R-menthadienol or a mixture of 1S, 4S-menthadienol and 1R, 4S-menthadienol is used.

Surprisingly, the process according to the invention allows the synthesis of enantiomerically pure (+) -cannabinoids and derivatives thereof from diastereomerically pure starting materials, i.e. mixtures of 1S, 4S-menthadienol with 1R, 4S-menthadienol. The final product can be purified without any time consuming, expensive chiral purification process and still achieve > 99% purity and > 99% enantiomeric purity.

According to a preferred embodiment of the above process, step i) is carried out in a halogen-free solvent, preferably toluene. Other possible solvents are benzene, xylene, cyclohexane or methyl tert-butyl ester.

Advantageously, the process according to the invention can be carried out in halogen-free solvents, which avoids ecological problems. Preferably, step i) is performed in toluene, which provides the best efficiency and reaction yield.

According to a further preferred embodiment of the above process, a solution of a lewis acid catalyst is provided in step i) and contacted with a solution of the compound of formula (II) and 4S-menthadienol.

In order to ensure that the catalyst is always present in the reaction chamber, a solution of the catalyst is charged into the reaction chamber, and then a solution of the compound of formula (II) and 4S-menthadienol is charged into the reaction chamber. In a continuous flow reaction process in which both solutions are pumped through a continuous flow reactor, the solution of the catalyst preferably begins before and ends after the solution of the compound of formula (II) and 4S-menthadienol.

In a preferred embodiment, the lewis catalyst is boron trifluoride etherate. The use of boron trifluoride etherate as a catalyst has been shown to provide a highly efficient reaction with good yields.

As mentioned above, any allyl alcohol can be used as reaction partner for the transesterification reaction described in step ii). Branched or unbranched, saturated or unsaturated alkyl alcohols and cyclic alkyl alcohols may be suitable. However, the best yields and selectivities are obtained when using unbranched, saturated alkyl alcohols having one primary hydroxyl group.

In a further preferred embodiment, the transesterification with ethylene glycol and/or 1, 2-pentanediol is carried out in step ii).

Transesterification of compound (1) with ethylene glycol and/or 1, 2-pentanediol in step ii) provides said compound (2) and/or compound (4), respectively. Compound (2) and compound (4) have been shown to have biological activity, which allows therapeutic applications as explained in more detail below. Furthermore, they are easily decarboxylated to give compound (3).

Scheme 2: transesterification of Compound (1) with ethylene glycol (top) and 1, 2-pentanediol (bottom)

In another preferred embodiment, an acid is used in step ii). The decarboxylation reaction may be carried out by adding an acid, such as sulfuric acid. Alternatively, hydrochloric acid may be used, but sulfuric acid is preferably used.

Scheme 3: transesterification with ethylene glycol and decarboxylation

The crude (+) -cannabinoids can be purified by flash chromatography or thin layer distillation. The purification step may be followed by a final crystallization step which yields (+) -cannabinoid with a purity of > 99% and (+) -cannabinoid with absolute enantiomeric purity, respectively.

According to another aspect, the invention relates to a (+) -cannabinoid compound as described above for use in a method of treatment, in particular for use in the treatment and/or prophylaxis of a medical condition. The therapeutic effect of (+) -cannabinoid compounds may be attributed to their association with cannabinoid receptor CB ₁ And CB ₂ Is described in (a) and (b) interact with each other.

Mammalian tissue comprises at least two cannabinoid receptors CB ₁ And CB ₂ Both coupled to G protein. CB (CB) ₁ Receptors are predominantly expressed by neurons of the central and peripheral nervous systems, whereas CB ₂ Receptors are found in the center and periphery of certain non-neuronal tissues, particularly immune cells (Pertwee et al, 2010). The presence of endogenous ligands for cannabinoid receptors has also been demonstrated (Pacher et al, 2006). The discovery of this "endocannabinoid system" has prompted the development of a range of novel cannabinoid receptor agonists and antagonists, including the discovery of CB ₁ Or CB ₂ Receptors are significantly selective for several species.

CB ₁ Receptors are located in the central and peripheral nervous systems and reside in one orthosteric and several allosteric binding sites for potential ligand binding (Price et al, 2005; adam et al, 2007; horswill et al, 2007; navarro et al, 2009). Because it is distributed throughout the nervous system, CB ₁ Activation of receptors affects various cognitive processes (such as attention, memory, motor function and pain perception) (Pertwe et al, 2008; elphick et al, 2001). Through CB ₁ Antagonists inhibit CB ₁ Receptors provide extended drug applicability to target treatment of, for example, obesity (dorish et al, 2008), opioid abuse (Sharma et al, 2007), and parkinson's disease (Concannon et al, 2015).

CB ₂ Receptors are located mainly in peripheral tissues of the immune and gastrointestinal systems, but can also be found in neurons of the brain (Chen et al, 2017). Thus, CB ₂ The receptor promotes anti-inflammatory and immunomodulating (immunosuppression),Induction of apoptosis, induction of cell migration), therapeutic effect (Basu et al, 2011). In addition, CB ₂ Receptors have potential therapeutic roles in the treatment of neurodegenerative diseases such as Alzheimer's disease (Benito et al, 2003).

Both central and peripheral cannabinoid receptors are members of the seven-helix domain G protein-coupled receptor superfamily. Cannabinoid receptors are known to mediate their effects by inhibiting adenylate cyclase by pertussis toxin-sensitive Gi/o (Pacher et al, 2006).

For use in medical/pharmaceutical formulations, it is necessary to target different actions by selective interactions of the substances with CB receptors. For example, 9-THC is a non-selective CB ₁ And CB ₂ Agonists, therefore, their efficacy is not clear.

All tested (+) -cannabinoids showed a binding to CB in the nanomolar range ₁ And CB ₂ This contributes to the potential use of these compounds at therapeutic doses.

Surprisingly, none of the compounds showed CB ₁ Agonist activity, but all compounds showed CB ₁ Antagonistic activity. (+) -CBD shows CB ₁ Antagonistic activity, additionally shows CB ₂ Agonist activity. (+) -CBD-HPE pair CB ₁ And CB ₂ Exhibit the same affinity, is a CB with a margin ₂ Very potent CBs of agonist activity ₁ Antagonists.

The test compounds have different but distinct activity profiles, with (+) -CBD being the most active. The selective activity profile enables the title cannabinoids to be used for different targets depending on the indication. Particularly surprising is the CB ₁ Antagonism provides an opportunity for targeted treatment of a variety of indications, from obesity, opioid abuse to neurodegenerative diseases, such as parkinson's disease.

Primary human monocytes are one type of white blood cells, or white blood cells (PBMCs: peripheral blood mononuclear cells), present in human blood, representing about 10% to 30% of the PBMCs. Monocytes and their macrophages and dendritic cell progenies in the immune system, phagocytosis, and anti-tumor Three major and critical roles are played in pro-presentation and cytokine production. Monocytes also affect the process of adaptive immunity as part of the innate immune system of vertebrates. Has been shown to express CB by human monocytes ₂ These receptors play an important role in immune function and immune regulation (Klein et al, 2003). To CB ₁ Studies of the receptor and its role in primary human monocytes have shown that activation of this receptor promotes the pro-inflammatory response of macrophages. Inhibition of CB ₁ And selectively activating CB ₂ Can inhibit the pro-inflammatory response of macrophages (Han et al 2009).

Primary human fibroblasts are a major source of extracellular matrix (ECM) proteins and play a critical role in determining cell phenotype and function in addition to providing scaffolds for cells. Fibroblasts contribute to the injury response during both the initiation and the lysis phases. They also play a role as helper cells in many immune and inflammatory responses. Fibroblasts are capable of producing or reacting to a variety of cytokines, and these mediators allow fibroblasts and leukocytes to cooperate in complex processes such as wound healing. Fibroblasts are able to alter the output of their extracellular matrix components in response to mediators released by other cell types. Some chronic inflammatory diseases in humans eventually progress to disabling fibrotic diseases, showing how continued activation of the immune system leads to serious disorders of fibroblast function. Studies have shown CB ₂ The receptor can regulate fibrosis in repair of skin wounds in mice. Previous studies have shown that CB is detected in the skin of mice ₂ Receptors are expressed dynamically in neutrophils, macrophages and myofibroblasts during the healing process of mouse skin wounds (Zheng et al 2012). Research also shows that CB ₂ The agonist JWH-133 prevents the development of skin and lung fibrosis and reduces fibroblast proliferation and autoantibody development. For lack of CB ₂ Experiments performed on mice of (a) confirm CB ₂ Effects on systemic fibrosis and development of autoimmunity (Li et al, 2016; serrattaz et al, 2010). Except CB ₂ In addition to the regulation of fibroblasts, the CB ₁ Receptor(s)And is shown to be a similarly promising target. For example, in three chronic liver injury models, CB ₁ The antagonist SR141716A inhibited the progression of fibrosis and indicated CB ₁ Receptor antagonists have also brought promise for the treatment of liver fibrosis (Teixeira-Cletc et al, 2006; marquart et al, 2010).

Human HaCAT keratinocytes are the major cell type of the epidermis, the outermost layer of the skin, accounting for 90% of the skin cells. The primary function of keratinocytes is to form a barrier against environmental damage to the skin caused by pathogenic bacteria, fungi, parasites, viruses, heat, ultraviolet radiation and moisture loss. Human keratinocytes have been shown to be involved in the peripheral endogenous cannabinoid system and to display CB ₁ The signaling mechanisms of the receptors, which may be of importance for epidermal differentiation and skin development. Cannabinoids pass through non-CB ₁ /CB ₂ The mechanism inhibits human keratinocyte proliferation, and has potential therapeutic value in the treatment of psoriasis (De Petrocellis et al, 2004; wilkinson et al, 2007).

The (+) -cannabinoids tested exhibited different biological activities in different cell lines. Interestingly, all cannabinoids have different effects. In most cases, this may be with their CB ₁ /CB ₂ Activity is related.

(+) -CBD has potent anti-inflammatory effects on monocytes (except IL-1, MMP9 and isoprostane) and fibroblasts. These effects are likely to be associated with the CB of the compound ₁ antagonism/CB ₂ Agonism is associated.

(+) -CBDV has potent anti-inflammatory effects comparable to (+) -CBD, which is slightly less potent but also weakly inhibits LPS-induced IL-1. This effect is partially associated with CB of a compound ₁ Antagonism is relevant. Since (+) -CBDV does not show CB ₂ Agonism, it can therefore be speculated whether its anti-inflammatory effect is strictly due to CB ₁ Antagonism or the presence of additional interactions.

(+) -CBD-ME showed only slight inhibition of LPS-mediated TNF-alpha and IL-6, less inhibition of IL-1-induced PGE2 of fibroblasts, and Poly The inhibition of TIMP1 by C-stimulated HaCat was the least active compound of the 5 subjects. This directly corresponds to its pair CB ₁ /CB ₂ The weak action of the receptor ((+) -CBD-ME shows the weakest CB of the test compound ₁ Antagonism of CB ₂ No effect on the receptor).

(+) -CBD-GE also showed strong inhibition in monocytes and fibroblasts, which prevented most of the inflammation-inducing parameters other than IL-8 and MMP9, which were even increased by the cannabinoid. This increase may be associated with the cytotoxic effects of the high dose compounds. Weak inhibition of MMP9 and TIMP1 was observed in PolyI:C treated HaCat cells.

(+) -CBD-HPE is not as potent as (+) -CBD, (+) -CBDV and (+) -CBD-GE, and has potent inhibitory effects on PGE2 of LPS-induced monocytes, IL-6 and IL-8 of IL-1 stimulated fibroblasts, and MMP9 and TIMP of Poly-I: C-induced HaCat cells. This is surprising because (+) -CBD-HPE shows the highest CB ₁ Antagonism, and is the only one that exhibits CB in addition to (+) -CBD ₂ Other cannabinoids of agonism. Of all 5 cannabinoids tested, (+) -CBD-HPE showed the lowest Ki values in terms of binding to both cannabinoid receptors. Additional interactions may contribute to the effects shown.

The invention therefore also relates to a compound of formula (I) or a salt of a compound of formula (I), preferably selected from the group consisting of compounds (1) to (5) or salts thereof, for use in a method of treatment for achieving an effect selected from the group consisting of:

-anti-inflammatory,

Immunomodulation, immunomodulation,

Immunosuppression (S),

Immunostimulation(s),

-analgesia.

All (+) -cannabinoid compounds tested showed anti-inflammatory effects, although (+) -CBD, (+) -CBDV and (+) -CBD-GE were the most effective and therefore preferred therapeutic methods for achieving anti-inflammatory effects.

Due to CB ₂ The receptor agonist promotes an immunomodulatory effect,i.e. immunosuppression or immunostimulation, thus (+) -CBD and (+) -CBD-HPE are preferred for therapeutic methods that achieve immunomodulation, in particular immunosuppression or immunostimulation. Since (+) -CBD shows the strongest CB ₂ Agonism, it is therefore particularly preferred for therapeutic methods to achieve immunomodulation, in particular immunosuppression or immunostimulation. CB (CB) ₂ Receptor agonists may also be used as analgesic therapies. Thus, (+) -CBD and (+) -CBD-HPE, in particular (+) -CBD, are preferred therapeutic methods for achieving analgesic effects.

Thus, a particularly preferred embodiment of the present invention is a compound of formula (I) for use in the above-described method of treatment, wherein the compound of formula (I) is compound (3) or a salt of compound (3).

CB 2-agonists are promising analgesic therapies for the treatment of various painful conditions (chronic, neuropathic, inflammatory) (Le Boisselier et al, 2017; likar et al, 2017). In addition, CB 2-agonists are useful in the treatment of neuroinflammatory and neurodegenerative diseases, such as multiple sclerosis (Pertwe, 2007; dittel, 2008), huntington's disease (Sagredo et al 2012), alzheimer's disease (Aso et al 2016), and in the treatment of cerebral stroke (Zhang et al 2007).

CB 2-agonists are also effective methods of treating peripheral inflammatory diseases such as arteriosclerosis (Mach et al, 2008) or inflammatory bowel disease (Izzo et al, 2008; wright et al, 2008), ischemia (ba tkai et al, 2007), diabetic nephropathy (baritta et al, 2011) and cirrhosis (Izzo et al, 2008; mallat et al, 2007; lotersztajn et al, 2008).

Epidemiological and preclinical data indicate that CB is activated ₂ The receptor has beneficial therapeutic effects on osteoporosis (Ofek et al, 2006). In addition, clinical data indicate that CB ₂ Agonists are effective against certain cancer types (Izzo et al, 2008; wright et al, 2008; guzman, 2003).

CB1 antagonists are promising therapies for the treatment of obesity and thus diabetes (Dourish et al, 2008; badal et al, 2017; wagner et al, 2012) and are therefore considered important for the pattern of weight loss. In addition, they show beneficial effects in the treatment of drug withdrawal and drug withdrawal (alcohol, tobacco, narcotics) (Ravan et al, 2014; chandler et al, 2009; koob et al, 2014).

CB1 antagonists may also treat nonalcoholic fatty liver (NAFLD) by blocking fatty liver metabolism (Badal et al, 2017). In addition, CB1 antagonists are promising therapies for the treatment of neurodegenerative diseases such as Parkinson's disease (Brotchie, 2003; cerri et al, 2014).

Accordingly, a preferred embodiment of the present invention is a compound of formula (I) or a salt of a compound of formula (I), preferably selected from the group consisting of compounds (1) to (5) or salts thereof, for use in the treatment and/or prophylaxis: inflammatory diseases such as neuroinflammatory diseases, arteriosclerosis, inflammatory bowel disease or ischemia; and/or painful diseases such as chronic pain, neuropathic pain or diabetic neuropathy; and/or neurodegenerative diseases such as multiple sclerosis, huntington's disease, alzheimer's disease or parkinson's disease; and/or cerebral stroke and/or cirrhosis and/or osteoporosis and/or cancer and/or obesity and/or diabetes and/or liver fibrosis and/or nonalcoholic fatty liver disease and/or psoriasis, and/or for the treatment of narcotics, opioids, tobacco or alcohol withdrawal symptoms.

Preferably, the compound of formula (I) for use in the above-mentioned treatment and/or prophylaxis is compound (3) or a salt of compound (3).

The invention also relates to a pharmaceutical composition comprising one or more compounds of formula (I) or one or more salts of compounds of formula (I), preferably selected from the group consisting of compounds (1) to (5) or salts thereof.

The pharmaceutical composition according to the invention is preferably selected from the group consisting of solid galenic forms (e.g. tablets, coated or uncoated, with or without modified release), dragees (coated or uncoated, with or without modified release), capsules (soft or hard gelatine capsules, with or without modified release), granules (with or without modified release), powders (with or without modified release, e.g. nasal powders, ear powders), suppositories (coated or uncoated, with or without modified release), lozenges, chewing gums, semi-solid forms (e.g. hydrophobic ointments, such as hydrocarbon gels, liposomal gels, silicone gels, oleogels, and water-soluble forms such as ointments, such as absorption matrices, hydrophilic ointments, hydrophilic gels (hydrogels) or pastes, also nasal ointments), inhalants (e.g. pressurized gas metered dose inhalers, powder inhalers, nebulized inhalers, inhalable inhalation concentrates), injectables and implants (e.g. based on or as liquid or solid forms, which are suitable for the preparation of injectable solutions or solid matrices capable of modified release or as such), injectable solutions or solid matrices, active ingredient containing the active ingredient, patches.

Liquid forms are, for example, solutions, suspensions, emulsions, syrups (commonly known as cough syrups), mouthwashes, mouth rinses, throat sprays or nasal sprays, nose drops, nasal washes, ear drops, ear sprays and ear washes.

The pharmaceutical composition preferably comprises one or more components selected from the group consisting of: filling materials (e.g. cellulose, calcium carbonate), flow and anti-caking agents (e.g. talc, magnesium stearate), coatings (e.g. polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate), disintegrants (e.g. starch, crosslinked polyvinylpyrrolidone), plasticizers (e.g. triethyl citrate, dibutyl phthalate), substances for granulation (lactose, gelatin), retarders (e.g. dispersed polymethyl methacrylate/ethyl/2-trimethylaminoethyl ester copolymer, vinyl acetate/crotonic acid copolymer), compacting agents (e.g. microcrystalline cellulose, lactose), solvents, suspensions or dispersants (e.g. water, ethanol), emulsifiers (e.g. cetyl alcohol, lecithin), substances for changing rheological properties (silica, sodium alginate), substances for microbiological stabilization (e.g. benzalkonium chloride, potassium sorbate), preservatives and antioxidants (e.g. DL-alpha-tocopherol, ascorbic acid), substances for changing pH (lactic acid, citric acid), propellants or inert gases (e.g. fluorinated hydrocarbons, carbon dioxide), colorants (ferric oxide, titanium oxide, paraffin wax, such as in particular as may be found in the literature, Schmidt，P.C.，Christin，I.，，Wirk-und Hilfsstoffe für Rezeptur，Defektur und Groβherstellung“，1999，Wissenschaftliche Verlagsgesellschaft mbH Stuttgart or Bauer，K.H.，K-H.，Führer，C.，，Lehrbuch der Pharmazeutischen Technologie“，8.Auflage，2006，Wissenschaftliche Verlagsgesellschaft mbH Stuttgart)。

The preferred amounts of one or more compounds of formula (I) and/or one or more salts thereof in a pharmaceutical composition can be readily determined by a simple trial and error method by a person skilled in the art depending on the kind and use of the respective formulation.

Drawings

FIG. 1 shows the CB of the (+) -CBD as assessed in example 4 ₁ (left) and CB ₂ (right) binding.

FIG. 2 shows the CB of (+) -CBDV evaluated in example 4 ₁ (left) and CB ₂ (right) binding.

FIG. 3 shows the CB of the (+) -CBD-ME as evaluated in example 4 ₁ (left) and CB ₂ (right) binding.

FIG. 4 shows the CB of (+) -CBD-GE as assessed in example 4 ₁ (left) and CB ₂ (right) binding.

FIG. 5 shows the CB of the (+) -CBD-HPE as evaluated in example 4 ₁ (left) and CB ₂ (right) binding.

FIG. 6 shows the (+) -CBD pair CB evaluated in example 5 ₁ Influence of functional Activity (agonist Activity).

FIG. 7 shows the (+) -CBDV pair CB evaluated in example 5 ₁ Influence of functional Activity (agonist Activity).

FIG. 8 shows the (+) -CBD-ME pair CB evaluated in example 5 ₁ Influence of functional Activity (agonist Activity).

FIG. 9 shows the (+) -CBD-GE pair CB evaluated as in example 5 ₁ Influence of functional Activity (agonist Activity). (+) -CBD-GE showed cytotoxicity at 25. Mu.M.

FIG. 10 shows the (+) -CBD-HPE pair CB evaluated in example 5 ₁ Influence of functional Activity (agonist Activity).

FIG. 11 shows the (+) -CBD pair CB evaluated in example 5 ₁ Influence of functional Activity (antagonistic Activity).

FIG. 12 shows the (+) -CBDV pair CB evaluated in example 5 ₁ Influence of functional Activity (antagonistic Activity).

FIG. 13 shows the (+) -CBD-ME pair CB evaluated in example 5 ₁ Influence of functional Activity (antagonistic Activity).

FIG. 14 shows the (+) -CBD-GE pair CB evaluated as in example 5 ₁ Influence of functional Activity (antagonistic Activity). CBD-GE showed cytotoxicity at 25. Mu.M.

FIG. 15 shows the (+) -CBD-HPE pair CB evaluated in example 5 ₁ Influence of functional Activity (antagonistic Activity).

FIG. 16 shows the (+) -CBD pair CB evaluated in example 5 ₂ Influence of functional Activity (agonist Activity).

FIG. 17 shows the (+) -CBDV pair CB evaluated in example 5 ₂ Influence of functional Activity (agonist Activity).

FIG. 18 shows the (+) -CBD-ME pair CB evaluated in example 5 ₂ Influence of functional Activity (agonist Activity).

FIG. 19 shows the (+) -CBD-GE pair CB evaluated in example 5 ₂ Influence of functional Activity (agonist Activity).

FIG. 20 shows the (+) -CBD-HPE pair CB evaluated in example 5 ₂ Influence of functional Activity (agonist Activity).

FIG. 21 shows the (+) -CBD pair CB evaluated in example 5 ₂ Influence of functional Activity (antagonistic Activity).

FIG. 22 shows the (+) -CBDV pair CB evaluated in example 5 ₂ Influence of functional Activity (antagonistic Activity).

FIG. 23 shows the (+) -CBD-ME pair CB evaluated in example 5 ₂ Influence of functional Activity (antagonistic Activity).

FIG. 24 shows, for example(+) -CBD-GE pair CB evaluated in example 5 ₂ Influence of functional Activity (antagonistic Activity).

FIG. 25 shows the (+) -CBD-HPE pair CB evaluated in example 5 ₂ Influence of functional Activity (antagonistic Activity).

FIG. 26 shows the effect of (+) -CBD on cell viability of human monocytes as assessed in example 6.

FIG. 27 shows the effect of (+) -CBDV on the cell viability of human monocytes as assessed in example 6.

FIG. 28 shows the effect of (+) -CBD-ME on the cell viability of human monocytes as assessed in example 6.

FIG. 29 shows the effect of (+) -CBD-GE on the cell viability of human monocytes as assessed in example 6.

FIG. 30 shows the effect of (+) -CBD-HPE on cell viability in human monocytes as assessed in example 6.

FIG. 31 shows the effect of (+) -CBD on LPS-treated human monocyte inflammation parameters as assessed in example 6. Columns for each concentration are shown from left to right: IL1 beta, TNF alpha, IL6, IL8, MMP9, PGE2 and isoprostadin.

FIG. 32 shows the effect of (+) -CBDV on LPS-treated human monocyte inflammation parameters as assessed in example 6. Columns for each concentration are shown from left to right: IL1 beta, TNF alpha, IL6, IL8, MMP9, PGE2 and isoprostadin.

FIG. 33 shows the effect of (+) -CBD-ME on LPS-treated human monocyte inflammation parameters as assessed in example 6. Columns for each concentration are shown from left to right: IL1 beta, TNF alpha, IL6, IL8, MMP9, PGE2 and isoprostadin.

FIG. 34 shows the effect of (+) -CBD-GE on LPS-treated human monocyte inflammation parameters as assessed in example 6. Columns for each concentration are shown from left to right: IL1 beta, TNF alpha, IL6, IL8, MMP9, PGE2 and isoprostadin.

FIG. 35 shows the effect of (+) -CBD-HPE on LPS-treated human monocyte inflammation parameters as assessed in example 6. Columns for each concentration are shown from left to right: IL1 beta, TNF alpha, IL6, IL8, MMP9, PGE2 and isoprostadin.

FIG. 36 shows the effect of (+) -CBD on IL-1 treated human skin fibroblast inflammation parameters as assessed in example 6. Columns for each concentration are shown from left to right: IL6, IL8 and PGE2.

FIG. 37 shows the effect of (+) -CBDV on the inflammatory parameters of IL-1 treated human skin fibroblasts as assessed in example 6. Columns for each concentration are shown from left to right: IL6, IL8 and PGE2.

FIG. 38 shows the effect of (+) -CBD-ME on IL-1 treated human skin fibroblast inflammation parameters as assessed in example 6. Columns for each concentration are shown from left to right: IL6, IL8 and PGE2.

FIG. 39 shows the effect of (+) -CBD-GE on IL-1 treated human skin fibroblast inflammation parameters as assessed in example 6. Columns for each concentration are shown from left to right: IL6, IL8 and PGE2.

FIG. 40 shows the effect of (+) -CBD-HPE on IL-1 treated human skin fibroblast inflammation parameters as assessed in example 6. Columns for each concentration are shown from left to right: IL6, IL8 and PGE2.

FIG. 41 shows the effect of (+) -CBD on MMP1, MMP9 and TIMP1 in PolyIC treated human keratinocytes (HaCat) as assessed in example 6. Columns for each concentration are shown from left to right: MMP9, TIMP1 and MMP1.

FIG. 42 shows the effect of (+) -CBDV on MMP1, MMP9 and TIMP1 in PolyIC-treated human keratinocytes (HaCat) as assessed in example 6. Columns for each concentration are shown from left to right: MMP9, TIMP1 and MMP1.

FIG. 43 shows the effect of (+) -CBD-ME on MMP1, MMP9 and TIMP1 in PolyIC treated human keratinocytes (HaCat) as assessed in example 6. Columns for each concentration are shown from left to right: MMP9, TIMP1 and MMPl.

FIG. 44 shows the effect of (+) -CBD-GE on MMP1, MMP9 and TIMP1 in PolyIC-treated human keratinocytes (HaCat) as assessed in example 6. Columns for each concentration are shown from left to right: MMP9, TIMP1 and MMP1.

FIG. 45 shows the effect of (+) -CBD-HPE on MMPl, MMP9 and TIMP1 in PolyIC treated human keratinocytes (HaCat) as assessed in example 6. Columns for each concentration are shown from left to right: MMP9, TIMP1 and MMP1.

The present invention will be explained in more detail based on the following examples.

Example 1: synthesis of (+) -CBD (3)

Scheme 4: synthesis of (+) -CBD-ME (1)

71.4g (300 mMol) of methyl olivate and 50g (330 mMol) of 1R, 4S-menthadienol were dissolved together with toluene to achieve a combined volume of 400 ml- > solution A. 21.3g (150 mMol) of BF ₃ The diethyl ether complex was dissolved with toluene to a volume of 300 ml- > solution B. The two reaction solutions were then passed through two separate pump systems and a continuous flow reactor (rotation: 1200U/min, solution A:24ml/min, solution B:12 ml/min). Solution B starts before solution a and ends after solution a to ensure that the catalyst is always present in the reaction chamber. The reaction mixture was continuously collected in a vessel filled with 700ml of saturated NaHCO ₃ The solution was placed in a 2 liter laboratory reactor (30 ℃ C. Hob temperature, 300 rpm). Discarding the aqueous solution; the organic solution was washed 4 times with 250mL of 1% NaOH solution at 45 degrees. After washing, the organic solution was evaporated to dryness to give 94.58g of crude (+) -CBD methyl ester (purity=78%, yield 68%). The starting compounds can be used further without purification.

Exemplary purification (+) -CBD ME (1):

the crude product was purified by flash chromatography (eluent system cyclohexane/ethyl acetate=40/1 v/v). GC purity: 99.1%. Chiral GC analysis: enantiomeric excess was 99% (for enantiomerically pure starting materials). ¹ H NMR(400MHz，CDCl ₃ )δ11.98(s，1H)，6.50(s，1H)，6.21(s，1H)，5.55(s，1H)，4.52(p，J＝2.4，1.4Hz，1H)，4.41-4.36(m，1H)，4.16-4.06(m，1H)，3.90(s，3H)，2.89-2.79(m，1H)，2.78-2.69(m，1H)，2.44-2.33(m，1H)，2.29-2.15(m，1H)，2.09(dq，J＝17.9，4.0，2.5Hz，1H)，1.84-1.76(m，2H)，1.80-1.77(m，3H)，1.72-1.68(m，3H)，1.57-1.46(m，2H)，1.38-1.28(m，4H)，0.89(t，J＝6.8Hz，3H). ¹³ C NMR(101MHz，CDCl3)δ172.62，163.11，159.91，147.23，145.94，140.19，124.06，114.38，111.49，111.23，103.91，51.73，46.66，36.83，35.40，32.10，31.14，30.24，27.84，26.92，23.71，22.55，18.83，14.10。

Scheme 5: synthesis of (+) -CBD (3) via (+) -CBD GE (2)

49.2g (103 mMol) (+) -CBD-ME (1) were dissolved in 250mL ethylene glycol at 60 degrees and poured into a 1L laboratory reactor. 5.7g of potassium hydroxide were added and heating of the reaction mixture was started while stirring to 120℃and a vacuum of 500 mbar. The accumulated volatile by-products are distilled off. After 2 hours, the reaction temperature was raised to 150 degrees, which was maintained for an additional 3 hours. After 400mL of water and 130mL of n-heptane were added, the reaction mixture was cooled to 80 ℃. The temperature was further reduced to 40 degrees as 1.2mL sulfuric acid (50%) was slowly added until the pH was about 6. The layers were separated and the organic layer was washed once with 250mL of water and 250mL of sodium hydroxide solution (0.05%). Organic layer on Na ₂ SO ₄ Drying, and evaporating to dryness. Yield: 30.3g, GC purity: 53%.

Exemplary isolation and purification (+) -CBD GE (2):

after 2 hours at 120 degrees, a sample of the reaction mixture was taken, quenched with n-heptane and water, and neutralized with sulfuric acid (10% w/w). The layers were separated and the organic layer was evaporated to dryness. The crude (+) -CBD-GE (2) was purified by flash chromatography (eluent system n-heptane/ethyl acetate=4/1 v/v). GC purity: 97.8%. Chiral GC analysis: enantiomeric excess was 99% (enantiomerically pure starting material). ¹ H NMR(400MHz，CDCl ₃ )δ11.88(s，1H)，6.53(s，1H)，6.23(s，1H)，5.55(s，1H)，4.54-4.50(m，1H)，4.50-4.44(m，2H)，4.40-4.36(m，1H)，4.14-4.06(m，1H)，3.98-3.92(m，2H)，2.88(ddd，J＝13.1，8.8，6.7Hz，1H)，2.78(ddd，J＝13.1，8.7，6.8Hz，1H)，2.39(q，J＝8.1Hz，1H)，2.29-2.16(m，1H)，2.10(dq，J＝17.9，3.6Hz，1H)，1.84-1.76(m，2H)，1.80-1.77(m，3H)，1.71(s，3H)，1.60-1.49(m，2H)，1.36-1.29(m，4H)，0.89(t，J＝6.8Hz，3H). ¹³ C NMR(101MHz，CDCl ₃ )δ172.39，163.27，160.12，147.20，145.86，140.31，123.97，114.52，111.68，111.28，103.71，66.73，61.19，46.65，46.01，36.98，35.39，32.06，31.48，30.24，27.83，23.72，22.68，18.82，14.09。

Purification of (+) -CBD (3):

in this example, the crude (+) -CBD was purified by flash chromatography (eluent system cyclohexane/ethyl acetate = 20/1 v/v). After crystallization from n-heptane, flash chromatography can be replaced by thin layer distillation. GC purity: 99.8%. Chiral GC analysis: enantiomeric excess was 99% (for enantiomerically pure starting materials and starting materials containing up to 5% of the 4R-menthol enantiomer). ¹ H NMR(400MHz，CDCl ₃ )δ6.35-6.09(m，2H)，5.97(s，1H)，5.57(dt，J＝2.8，1.6Hz，1H)，4.66(p，J＝1.6Hz，2H)，4.56(d，J＝2.0Hz，1H)，3.85(ddp，J＝10.7，4.5，2.3Hz，1H)，2.48-2.41(m，2H)，2.38(ddd，J＝10.6，3.7Hz，1H)，2.30-2.17(m，1H)，2.09(ddt，J＝17.9，5.1，2.4Hz，1H)，1.88-1.81(m，1H)，1.79(dt，J＝2.6，1.2Hz，3H)，1.78-1.72(m，1H)，1.65(t，J＝1.1Hz，3H)，1.62-1.50(m，2H)，1.37-1.22(m，4H)，0.88(t，J＝7.0Hz，3H).13C NMR(101MHz，CDCl3)δ156.07，153.90，149.41，143.06，140.07，124.12，113.76，110.84，109.84，108.00，7.35，77.03，76.71，46.15，37.28，35.48，31.50，30.64，30.41，28.41，23.69，22.55，20.54，14.05。

Example 2: synthesis of (+) -CBD HPE (4)

Scheme 6: synthesis of (+) -CBD HPE (4)

10g (24 mMol) (+) -CBD-ME (1) were dissolved in 250mL of 1, 2-pentanediol at 60 degrees and poured into a 1L laboratory reactor. 1.1g of potassium hydroxide was added and heating of the reaction mixture was started with stirring to 120℃and a vacuum of 500 mbar. The accumulated volatile by-products are distilled off. After 2 hours, after 400mL of water and 130mL of n-heptane were added, the reaction mixture was cooled to 80 degrees. The temperature was further reduced to room temperature and neutralized with sulfuric acid (10% w/w). The layers were separated and the organic layer was washed once with 250mL water over Na ₂ SO ₄ Drying, and evaporating to dryness. The crude product (+) -CBD-HPE (4) was purified by flash chromatography (eluent system cyclohexane/ethyl acetate=10/1 v/v). GC purity: 98%. Chiral GC analysis: enantiomeric excess was 99% (for enantiomerically pure starting materials). ¹ H NMR(400MHz，DMSO-d ₆ )δ11.61(s，1H)，9.89(s，1H)，6.20(s，1H)，5.11-5.05(m，1H)，4.91-4.82(m，1H)，4.46(d，J＝2.7Hz，1H)，4.42(dd，J＝2.8，1.5Hz，1H)，4.24-4.11(m，2H)，3.95-3.86(m，1H)，3.81-3.71(m，1H)，3.03(td，J＝11.4，10.9，3.0Hz，1H)，2.74(s，2H)，2.22-2.05(m，1H)，1.94(dd，J＝16.7，4.1Hz，1H)，1.76-1.63(m，2H)，1.61(t，J＝1.8Hz，3H)，1.58(s，3H)，1.53-1.34(m，6H)，1.33-1.26(m，4H)，0.89(t，J＝7.0Hz，3H)，0.86(t，J＝6.7Hz，3H). ¹³ C NMR(101MHz，DMSO-d ₆ )δ171.22，162.07，160.33，148.61，144.07，130.72，125.60，114.82，110.15，109.84，103.50，68.95，67.27，43.32，35.71，35.61，35.44，31.36，30.91，30.13，29.12，23.13，22.00，18.88，18.12，13.86，13。

Example 3: synthesis of (+) -CBDV (5)

Scheme 7: synthesis of (+) -CBDV ME

91g (430 mMol) divarin methyl ester and 66g (430 mMol) 4S-menthadienol were dissolved with toluene to achieve a combined volume of 610 ml- > solution A. 20g (140 mMol) of BF ₃ The ether complex was dissolved with toluene to a volume of 177 ml- > solution. The two reaction solutions were then passed through two separate pump systems and a continuous flow reactor (rotation: 1200U/min, solution A:93ml/min, solution B:29 ml/min). The reaction mixture was continuously collected in a 2 liter laboratory reactor (30 ℃ C. Hob temperature, 300 rpm) with 750ml of saturated NaHCO3 solution. Discarding the aqueous solution; the organic solution was washed 7 times with 250mL of 1% NaOH solution at 40 ℃. After washing, the organic solution was evaporated to dryness to give 117g of crude (+) -CBDV-ME (purity=81%, yield 70%).

The starting compounds can be used further without purification.

Scheme 8: synthesis of (+) -CBDV (5) via (+) -CBDV-GE

117g (275 mMol) (+) -CBDV-ME was dissolved in 150mL ethylene glycol at 60 degrees and poured into a 1L laboratory reactor. 30.8g (550 mMol) of potassium hydroxide were dissolved in 100ml of ethylene glycol and added to the stirred solution. Heating of the reaction mixture was started under stirring to 120℃and a vacuum of 500 mbar. The accumulated volatile by-products are distilled off. After 2 hours, the reaction temperature was raised to 150 degrees, which was maintained for an additional 3 hours. After adding 550mL of water and 200mL of n-heptane, the reaction mixture was cooled to 80 degrees. The temperature was further reduced to 40 degrees while 45g sulfuric acid (50%) was slowly added until the pH was about 6. The layers were separated and the aqueous layer extracted once with 200ml of MTBE. The organic layer was combined and evaporated to dryness to afford 99 g of crude product. 23 g of Synalox oil were added to the crude product and the resulting mixture was distilled off on a thin layer distillation apparatus. The resulting distillate (62 g, 83.4% product according to GC analysis) was then crystallized from n-heptane. The white crystals obtained were recrystallized from n-heptane again to give the pure product. Yield: 27g. GC purity: 99.6%. Chiral GC analysis: enantiomeric excess of 99% (pairStarting materials that are enantiomerically pure and starting materials that contain up to 5% of the enantiomer of 4R-menthol). ¹ H NMR(400MHz，CDCl ₃ )δ6.37-6.08(m，2H)，5.98(s，1H)，5.57(dt，J＝2.8，1.6Hz，1H)，4.71(s，1H)，4.66(p，J＝1.6Hz，1H)，4.55(d，J＝2.1Hz，1H)，3.85(ddq，J＝10.5，4.5，2.4Hz，1H)，2.46-2.38(m，2H)，2.45-2.34(m，1H)，2.30-2.17(m，1H)，2.09(ddt，J＝17.9，5.1，2.5Hz，1H)，1.87-1.81(m，1H)，1.79(dd，J＝2.8，1.5Hz，3H)，1.77-1.72(m，1H)，1.65(t，J＝1.2Hz，3H)，1.64-1.52(m，2H)，0.90(t，J＝7.3Hz，3H). ¹³ C NMR(101MHz，CDCl ₃ )δ156.08，153.82，149.37，142.78，1140.07，124.13，113.82，110.85，109.82，108.11，77.35，77.04，76.72，46.17，37.58，37.25，30.41，28.41，24.03，23.69，20.51，13.81。

_{1 2} Example 4: in vitro binding to CB and CB receptors

Assessment as CB by Competition study ₁ And/or CB ₂ A compound of the receptor ligand having the desired properties to determine the affinity (Ki value) of the compound of both receptors for the classical cannabinoid ligand. Competition studies were performed with transfection CB ₁ Or CB ₂ The membrane of the receptor. For stock solutions, the compounds were dissolved at a concentration of 50mM to 100mM and stored at-20 ℃.

Experimental procedure

From human CB ₁ Or CB ₂ The membranes of the receptor transfected cells (RBHCB 1M400UA and RBXCB2M400UA, respectively) were provided by Perkin-Elmer Life and Analytical Sciences (Boston, mass.). CB (CB) ₁ Or CB ₂ B of acceptor film _max And K _d The values are variable. The batch used shows the following B _max And K _d Value of CB ₁ The receptor membranes were 1.9pmol/mg membrane protein and 0.16nM, respectively, for CB ₂ The receptor membranes were 5.2pmol/mg membrane protein and 0.18nM, respectively. CB (CB) ₁ The protein concentration of the receptor membrane was 8.0mg/ml, while CB ₂ The protein concentration of the receptor membrane was 4.0mg/ml. Commercial dieBinding buffer (for CB ₁ Binding buffer: 50mM TrisCl, 5mM MgCl ₂ .H ₂ O, 2.5mM EDTA, 0.5mg/mL BSA and ph=7.4; for CB ₂ Binding buffer: 50mM TrisCl, 5mM MgCl ₂ .H ₂ 0. 2.5mM EGTA, 1mg/mL BSA and pH=7.5) was diluted (1:20). Radioligand [ ³ H]CP55940 (144 Ci/mmol; perkinelmer) at a concentration of 0.10nM, a final volume of 200. Mu.l for CB ₁ Combining; at a concentration of 0.15nM, a final volume of 600. Mu.l for CB ₂ And (5) combining. The 96-well plates and tubes required for the experiments were siliconized with Sigmacote (Sigma). Membranes were resuspended in the corresponding buffers and incubated with radioligand and each compound at 30 ℃ for 90 minutes. Nonspecific binding was determined with 10 μm WIN55212-2 and 100% binding of radioligand to the membrane was determined by incubation with the membrane without any compound. Filtering byThe filtration was performed using a Filtermat A GF/C filter pretreated with 0.05% polyethylenimine (Perkin-Elmer). After filtration, the filter was washed nine times with binding buffer, dried, and the scintillation sheet was melt-melted thereon (melitex ^TM A, perkin Elmer). Radioactivity was then quantified using a liquid scintillation spectrophotometer (Wallac MicroBeta Trilux, perkin-Elmer).

Results

Using transfection CB ₁ Or CB ₂ Cell membrane of receptor (HEK 293 EBNA) and ligand of radiation [ ³ H]-CP55940, evaluating the affinity of the novel compound with a radioligand displacement assay. The evaluation of the compounds was performed in two stages. The first stage involves a simple screen for each compound that is unique and high concentration (40 μm). Data were collected from at least three experiments, each performed in triplicate. The second stage selects only the permutations that can be replaced ³ H]-CP55940(CB ₁ 0.10nM, CB ₂ 0.15 nM) of those compounds that bind more than 50%. This is achieved by ³ H]-CP55940(CB ₁ 0.10nM, CB ₂ 0.15 nM) and various concentrations of the selected compound(10 ^-4 -10 ^-11 M) competition studies performed. Using GraphPadThe data were analyzed by version 5.01 (GraphPad software, san diego, california, usa) to calculate Ki values for at least three experiments performed in triplicate for each point, expressed as mean ± SEM. The calculated Ki values are shown in Table 2. The combined figures are shown in figures 1 to 5.

Table 2: CB of a Compound ₁ And CB ₂ Binding Activity

Compounds of formula (I)	CB 1 -Ki(nM)	CB 2 -Ki(nM)	CB 1 /CB 2 Selectivity of
				(+)-CBD	982	40.5	24.3
(+)-CBDV	294	33.1	8.9
				(+)-CBD-ME	345	28.0	12.3
(+)-CBD-GE	359	12.9	27.8
				(+)-CBD-HPE	3.1	0.8	3.9

Example 5: functional analysis of transfected cells

After determining the binding affinity of these compounds for the expression of cannabinoid receptors, their effect on cannabinoid receptors (agonism, antagonism) was analyzed.

Experimental procedure

HEK 293T-CB ₁ And HEK 293T-CB ₂ Cells (with CB) ₁ And CB ₂ Stable transfection of cDNAs) (10 ⁵ Per ml) were incubated in 24-well plates and transiently transfected with 0.5 μg,/ml of plasmid CRE-Luc containing 6 consensus CAMP Response Elements (CREs) linked to firefly luciferase. Transient transfection was performed using Rotifect (Carl Roth GmbH, karlsruhe, germany) according to the manufacturer's instructions and harvested 24 hours after transfection.

For CB ₁ Agonist activity, transfected cells were treated with increasing concentrations of test compound or WIN55, 212-2 (CB) ₁ Positive control) and then luciferase activity was measured in the cell lysate (1-2). Forskolin is an adenylate cyclase activator and is used at 10. Mu.M with CB ₁ Positive control of cAMP signaling pathway activated by receptor independent mechanisms.

For CB ₁ Antagonistic activity, CB ₁ Cells were pre-incubated with test compounds for 15 minutes and then stimulated with WIN55, 212-2 for 6 hours.

To measure CB ₂ Agonist activity, HEK293T-CB ₂ CRE-luc cells are treated with increasing concentrations of test compound or WIN55, 212-2 (CB ₂ Positive control) for 15 minutes and then treated with Forskolin (10 μm) over 6 hours.

For CB in cells ₂ Antagonistic activity potential inhibition of Forskolin-induced CRE-luc inhibition by compounds was analyzed. As positive controls, two known CBs of AM630 or SR144588 were used ₂ Antagonists.

After 6 hours of stimulation, cells were lysed (in 25mM Tris-phosphate, pH 7.8,8mM MgCl) as indicated by the luciferase assay kit (Promega, madison, wis.) ₂ 1mM DTT, 1% Triton X-100 and 7% glycerol) and luciferase activity was determined using Autolumat LB 9501 (Berthold Technologies, bad Wildbad, germany). The background value obtained with lysis buffer was subtracted from each experimental value and specific transactivation was expressed as double induction (CRE-Luc) above basal levels.

Results

CB ₁ Agonist activity

HEK 293T-CB ₁ Cells were transfected with CRE-Luc plasmid and stimulated with Win-55, 212-2 (1. Mu.M, positive control) or test compound for 6 hours after 24 hours. Negative controls (untreated cells, 0% activated) are not listed. The results indicated that none of the 5 test compounds showed CB ₁ Agonist activity (fig. 6 to 10).

CB ₁ Antagonistic activity

HEK 293T-CB ₁ Cells were transfected with CRE-Luc plasmid and stimulated with Win 55,212-2 (1. Mu.M positive control) for 6 hours after 24 hours with or without test compound. Negative controls (untreated cells, 0% activated) are not listed. The results indicate that all of the test compounds showed CB ₁ Antagonistic Activity (FIGS. 11 to 15), (+) -CBD-HPE was the strongest CB ₁ Antagonists (figure 15).

CB ₂ Agonist activity

HEK 293T-CB ₂ Transformation of cells with CRE-Luc plasmidStained and stimulated with forskolin (10 μm, positive control) in the presence or absence of WIN 55, 212-2 or test compound for 6 hours after 24 hours. Negative controls (untreated cells, 0% activated) are not listed. (+) -CBD (FIG. 16) and (+) -CBD-HPE (FIG. 20) showed CB at higher concentrations ₂ Agonist activity. This activity of the other test compounds was negative (fig. 17 to 19).

CB ₂ Antagonistic activity

The following studies on CB by the compound ₂ Potential antagonistic activity of the receptor. When two known CBs are used ₂ The presence of the antagonists AM630 or SR144588 prevented WIN 55212-2 inhibition in Forskolin-induced CRE-Luc inhibition. Negative controls (untreated cells, 0% activated) are not listed. The results show that none of the test compounds showed CB ₂ Antagonistic activity (fig. 21 to 25).

Table 3: CB of test Compound ₁ And CB ₂ Agonism/antagonism

Example 6: biological Activity Studies

Test compounds were dissolved in DMSO (10 mg/ml stock solution) and diluted in medium for the experiments described below.

Experimental procedure

Cell culture: human primary monocytes were extracted from whole blood of medical healthy volunteers from a local blood bank (university of Freiburg, germany) providing written informed consent, following a standardized protocol using complete endotoxin-free culture (gradient formulation, lymphocyte separation medium, PAN Biotech, P04-60125, germany Ai Dengba Hz). Using a 50ml tube, 25ml of Pancoll was filled with 25ml of blood (buffy coat). The gradient was established by centrifugation at 1800rpm for 40 minutes at 20℃under slow acceleration and deceleration. Intermittent peripheral blood mononuclear cells were carefully removed and resuspended in 50ml of pre-warmed Phosphate Buffered Saline (PBS) (Pan Biotech, P04 36500) and then centrifuged at 1600rpm for 10 minutes at 20 ℃. The supernatant was discarded, the pellet washed in 50ml PBS and centrifuged as described above. The pellet was then resuspended in 50ml of RPMI-1640 low endotoxin medium supplemented with 10% human serum (Hexcell, berlin, germany, SP 2080). After counting the cells in a particle counter (Euro Diagnostics, germany, g Lei Feier Germany), the cells were inoculated in 24-well plates for enzyme-linked immunosorbent assay (ELISA) (2.2 mio. Cells/well), or in 96-well plates at a density of 2X 104 cells/well for cell viability testing, and at 37℃with 5% CO ₂ And (5) incubating. The medium and non-adherent cells (lymphocytes) were removed and fresh RPMI-1640 medium containing 1% human serum was added. The enriched monocytes were then ready for the experiment. Primary human fibroblasts and HaCat keratinocytes were obtained from Uniklinik Freiburg. All cells were incubated at 37℃with 5% CO ₂ Is stored in a humidified atmosphere containing 10% FBS (Bio&Sell, feucht, germany) and 1% antibiotic penicillin/streptomycin (from Invitrogen, DMEM complete medium) in supplemented DMEM (Invitrogen, life technologies, dammstatt, germany) medium.

Cell viability: cells were incubated with cannabinoids (3 doses, n=4) for 24 hours. Cytotoxicity was analyzed by alamar blue staining (formazan). Cells were then washed once with 100 μl of PBS, and then 100 μl of medium-alma blue mixture (90% medium, 10% alma blue, DAL1025, thermo Fisher) was added to each well. Then humidifies 5% CO at 37 DEG C ₂ The plates were incubated for 2 hours and the colour response was determined using a 96-well plate reader (Berthold, ohinburgh, germany, excitation 544nm, emission 590 nm).

Determination of inflammatory molecules in fibroblasts: primary human fibroblasts were cultured as described above and seeded in 24-well plates (500000 cells/well). Cells were incubated with IL-1 β (Roche, mannham, germany, 10U/ml) for 24 hours in the absence or presence of cannabinoids (5 doses, n=3). Non-stimulated cells served as negative controls. 24 hours after cell stimulation, the supernatant was removed, centrifuged, and the concentrations of IL-6, PGE2 and IL-8 were studied using ELISAs according to the manufacturer's protocol (PGE 2 from Cayman/Biomol, hamburg, germany; IL-6 and IL-8 from Immunotools, freund's, germany). The respective extinction was determined using a 96-well plate reader (Berthold, ohinburgh, germany).

Determination of MMPs and TIMPs in keratinocytes: keratinocytes (HaCat) were cultured as described above. Cells were seeded in 24-well plates and incubated with PolyI: C (InvivoGen, san Diego, calif., U.S. for 24 hours in the absence or presence of cannabinoids (5 doses, n=3). Non-stimulated cells served as negative controls. 24 hours after cell stimulation, the supernatant was removed, centrifuged, and the concentrations of MMP1, MMP9, and TIMP1 were studied using ELISAs according to the manufacturer's protocol (from Biotechne, wisbaden, germany). The respective extinction was determined using a 96-well plate reader (Berthold, ohinburgh, germany).

Determination of cytokines and PGE2 in primary human monocytes: cells were incubated with LPS (Sigma Aldrich, germany Tao Fuji, 10 ng/ml) for 24 hours. Cannabinoids (5 doses) were added 30 minutes prior to LPS treatment. After 24 hours, the supernatant was removed, centrifuged, and the concentrations of IL-1beta, IL-8, IL-6, TNFalpha, MMP9, isoprostan and PGE2 were studied using the manufacturer's protocol (PGE 2 and isoprostan, from Cayman/Biomol, german Hamburg) or ELISAs (IL-1 beta, hiss, german Frieburg; TNFalpha, IL-6 and IL-8, immunotols, frasiothey, germany; MMP9, biotechne, wisbadn, germany). The respective extinction was determined using a 96-well plate reader (Berthold, ohinburgh, germany). Each dose was analyzed 4 times in two buffy coats of two different blood donors (2 buffy coats used from 2 different healthy blood donors, with a final n=4 values).

Results

Influence on the viability of human monocyte cells

Cytotoxicity assays were performed on primary human monocytes. (+) -CBD and (+) -CBDV started to affect cell viability at 25. Mu.M and higher (FIGS. 26 and 27), while the other three cannabinoids started to affect cell viability at 50. Mu.M (FIGS. 28 to 30). To be able to compare the activity of the five cannabinoids, 25 μm was used as the highest dose.

Effects on LPS-induced human monocyte inflammation parameters

As shown in FIG. 31, (+) -CBD started to significantly inhibit LPS-stimulated release of TNF alpha and PGE2 at 0.1. Mu.M (TNF alpha) and 1. Mu.M (PGE 2), with the strongest effect at 25. Mu.M, indicating an inhibition of about 80%. LPS-induced IL-6 was inhibited at doses of 5. Mu.M to 25. Mu.M, IL-8 was inhibited only at doses of 25. Mu.M, whereas LPS-mediated IL-1β, MMP9 and isoprostane were unaffected by (+) -CBD. The non-inhibitory effect on these 3 parameters indicated that the use of 25 μm did not kill cells and thus affected other parameters.

At the highest dose, (+) -CBDV showed comparable effects to (+) -CBD except for a weaker effect on TNFα and a slight effect on IL-1β (FIG. 32).

(+) -CBD-ME inhibited LPS-induced IL-6 and TNF. Alpha. Only slightly, with an inhibition of about 20%, but increased chemokine IL-8 at doses of 5. Mu.M to 25. Mu.M and IL-1 at doses of 25. Mu.M (FIG. 33).

As shown in FIG. 34, (+) -CBD-GE dose-dependently inhibited LPS-stimulated release of TNFα, IL-6, IL-1, isoprostane and PGE2 from 0.1. Mu.M, with the strongest effect at 25. Mu.M, indicating an inhibition of IL-6 and PGE2 of about 80% and an inhibition of about 40% for the other three parameters. LPS-induced IL-8 was not affected, whereas inhibition of LPS-mediated MMP9 was significantly enhanced (20% to 60% higher than LPS values) at doses of 5 μm to 25 μm.

(+) -CBD-HPE inhibited LPS-induced PGE2 only slightly at doses of 5. Mu.M to 25. Mu.M and IL-6 at doses of 10. Mu.M and 25. Mu.M. At a dose of 25. Mu.M, LPS-stimulated IL-8 and MMP9 were only slightly blocked, whereas LPS-mediated IL-1β inhibition was significantly increased (almost twice the LPS effect) at the 25. Mu.M dose, whereas LPS-induced TNFα was unaffected (FIG. 35).

Effects on IL-1-induced human skin fibroblast inflammation parameters

As shown in FIG. 36, (+) -CBD significantly and dose dependently inhibited IL-1 stimulated IL-6 and IL-8 release starting at 0.1. Mu.M, with the strongest effect at 25. Mu.M, indicating an inhibition of more than 90%. At doses of 10. Mu.M to 25. Mu.M, IL-1 induced PGE2 was strongly inhibited (inhibition exceeding 80%).

(+) -CBDV showed comparable situation to (+) -CBD, but with the initial use of 5. Mu.M, the activity towards IL-6 and IL-8 was weaker. IL-1-induced PGE2 was strongly inhibited (inhibition over 80%) at doses ranging from 10. Mu.M to 25. Mu.M (FIG. 37).

(+) -CBD-ME slightly increased LPS-induced IL-6 and IL-8 and slightly decreased IL-1-mediated PGE 2. The 25 μm dose appears to be toxic to fibroblasts (fig. 38).

As shown in FIG. 39, the parameter of dose-dependent inhibition of all IL-1 stimulation by (+) -CBD-GE from 5. Mu.M was the strongest with 25. Mu.M, indicating an inhibition of IL-6 and IL-8 of about 95% and an inhibition of P6E2 of about 80%.

(+) -CBD-HPE inhibited IL-1-induced IL-6 and IL-8 at doses of 10. Mu.M and 25. Mu.M (inhibition 95%) and PGE2 at doses of 25. Mu.M (inhibition 50%) (FIG. 40).

Effect on PolyI: C-induced proteases in human HaCat keratinocytes

As shown in FIG. 41, (+) -CBD slightly inhibited Poly I: C stimulated MMP9 and TIMP1 from 5. Mu.M, with 25. Mu.M acting most strongly, indicating an inhibition of MMP9 of about 40% and TIMP1 of about 70%. Poly I: C induced MMP1 was only slightly inhibited (greater than about 40% inhibition) at a dose of 25. Mu.M.

(+) -CBDV showed slight inhibition of PolyI: C-stimulated MMP9 and TIMP1 at a dose of 25. Mu.M, but enhanced PolyI: C-stimulated MMP1 (FIG. 42).

(+) -CBD-ME slightly reduced PolyI: C-induced TIMP1 (inhibition 40%) in a dose-dependent manner at doses of 5. Mu.M to 25. Mu.M, but enhanced PolyI: C-stimulated MMP9 and MMP1 (FIG. 43).

As shown in FIG. 44, (+) -CBD-GE (25. Mu.M) had slight inhibition (30% to 50%) of PolyI: C stimulated MMP9 and TIMP1, but had no effect on PolyI: C induced MMP 1.

(+) -CBD-HPE inhibited all parameters induced by Poly I: C, at a dose of 25. Mu.M, MMP9 was inhibited at 40%, TIMP1 was inhibited at 60% and MMP1 was inhibited at 20% (FIG. 45).

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Claims (8)

1. A process for the production of a compound of formula (I)

Or a salt of a compound of formula (I),

wherein x=h or-COOY,

wherein Y = a saturated or unsaturated, branched or unbranched alkyl, aryl or heteroaryl group, each having 1 to 12 carbon atoms, and being optionally substituted by one or more amino groups, hydroxyl groups and/or halogens, and

where n=2 or 4,

the method comprises the following steps:

i) Reacting 4S-menthadienol with a compound of formula (II),

where n=2 or 4,

to obtain a compound of formula (III),

where n=2 or 4,

wherein step i) is performed as a continuous flow reaction process, step i) is performed in a halogen-free solvent, step i) using a lewis acid catalyst;

optionally:

ii) transesterification of the compounds of formula (III), and/or

iii) Decarboxylation of the compound of formula (III).

2. The method of claim 1, wherein the compound of formula (I) is selected from the group consisting of compounds (1) to (5) or salts thereof

3. The method according to claim 1, wherein in step i) pure 1s,4 s-menthadienol or pure 1r,4 s-menthadienol or a mixture of 1s,4 s-menthadienol and 1r,4 s-menthadienol is used.

4. The process according to claim 1, wherein step i) is carried out in toluene.

5. The process according to claim 1, wherein in step i) a solution of a lewis acid catalyst is provided and contacted with a solution of the compound of formula (II) and 4S-menthadienol.

6. The process of claim 1, wherein the lewis acid catalyst is boron trifluoride etherate.

7. The process according to claim 1, wherein in step ii) a transesterification reaction with ethylene glycol and/or 1, 2-pentanediol is carried out.

8. The process according to claim 1, wherein an acid is used in step iii).